Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment

We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescentproteins ( FPs) from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non- fluorescentto fluorescent fatty acid-binding proteins(FABPs). Using transcriptomic analysis of two species of brightly fluorescent Kaupichthyseels ( Kaupichthyshyoproroides and Kaupichthysn. sp.), two new FPswere identified, cloned and characterized (Chlopsid FPI and Chlopsid FPII). We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPsdiverged as a protein family and are the sister groupto brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplicationevents. We show that fluorescentFABPs possess a unique, conserved tripeptideGly-Pro-Pro sequence motif, which is not found in non- fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FPfamily. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein’s fluorescentproperties. We present a phylogenetic reconstruction of this emerging FPfamily and describe additional fluorescentFABP members from groups of distantly related eels. The elucidation of this class of fish FPswith diverse properties provides new templates for the development of protein-based fluorescenttools. The evolutionary adaptation from fatty acid-binding proteinsto fluorescent fatty acid-binding proteinsraises intrigue as to the functional role of bright green fluorescencein this cryptic genus of reclusiveeels that inhabit a blue, nearly monochromatic, marine environment.